Toxicity typing using liver stem cells

• US 8,143,009 B2
• Filed: 06/01/2006
• Issued: 03/27/2012
• Est. Priority Date: 06/14/2000
• Status: Expired due to Term

• Current Assignee: VistaGen Therapeutics, Inc.
• Original Assignee: VistaGen, Inc.
• Inventors: Snodgrass, H. Ralph
• Primary Examiner(s): Marvich, Maria
• Application Number:
  US11/445,733
• Publication Number:
  US 20070111195A1
• Time in Patent Office: 2,126 Days
• Field of Search: None
• US Class Current: 435/6.13
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: G01N 33/5014 for testing toxicity

Abstract

This invention provides methods and systems for identifying and typing toxicity of chemical compositions, as well as for screening new compositions for toxicity. The invention involves detecting alterations in gene or protein expression and hence establishing molecular profiles in isolated mammalian LSCs contacted with various chemical compositions of known and unknown toxicities, and correlating the molecular profiles with toxicities of the chemical compositions.

Claims

1. A first pass method of predicting the type of toxicity of a chemical compound of unknown toxicity, the method comprising the steps of:

a) contacting an isolated population of mammalian liver stem cells (LSCs) with the chemical compound of unknown toxicity;

b) creating a molecular profile identifying the alterations in gene expression in the population of LSCs contacted with the chemical composition of unknown toxicity compared to LSCs in contact with culture medium only;

c) comparing the molecular profile of LSCs contacted with the chemical compound of unknown toxicity to sets of commonly altered RNAs identified from two or more molecular profiles of two or more isolated populations of LSCs wherein each isolated population of LSCs is contacted with one chemical composition of known toxicity, wherein the sets of commonly altered RNAs of the chemical compositions having known liver toxicity have been identified by

i) contacting each of the isolated populations of mammalian LSCs with one of the chemical compositions having known liver toxicity,

ii) creating molecular profiles identifying the alterations in gene expression for each of the population of LSCs contacted with each of the chemical compositions of known toxicity compared to LSCs in contact with culture medium only, and

iii) comparing molecular profiles for known chemical compounds of similar types of toxicity to identify sets of commonly altered RNAs for the chemical compounds of similar type of toxicity; and

d) predicting toxicity of the chemical composition of unknown toxicity wherein common alterations in gene expressions between the sets of commonly altered RNAs for chemical compounds of similar types of toxicity and the molecular profile from the LSCs contacted with the compound of unknown toxicity indicate the potential correlation of toxicity between the two.

2. The method of claim 1, wherein the level of gene expression is detected by a label directly or indirectly coupled to the nucleic acid.

3. The method of claim 2, wherein the label is selected from the group consisting of a fluorescent, a colorimetric, a radioactive, an enzyme, an enzyme substrate, a nucleoside analog, a magnetic, a glass, a latex bead, a colloidal gold, and an electronic transponder label.

4. The method of claim 2, wherein gene expression is detected by a nucleotide hybridization assay.

5. The method of claim 1, wherein the LSCs are of human origin.

6. The method of claim 5, further wherein the chemical compositions having known liver toxicities are selected from the group consisting of agents that are toxic to cells of one or more reproductive organs, teratogenic agents and carcinogens.

7. The method of claim 5, further wherein the chemical compositions having known liver toxicities are selected from the group consisting of agricultural chemicals, cosmetics, and environmental contaminants.

8. The method of claim 1, wherein the LSCs are of non-human mammalian origin.

9. The method of claim 8, wherein the non-human mammals are rodents.

10. The method of claim 8, further wherein the chemical compositions having known liver toxicities are selected from the group consisting of agents that are toxic to cells of one or more reproductive organs, teratogenic agents and carcinogens.

11. The method of claim 8, further wherein the chemical compositions having known liver toxicities are selected from the group consisting of agricultural chemicals, cosmetics, and environmental contaminants.

12. A method of selecting chemical compositions for liver toxicity preclinical animal studies, said method comprising

a) predicting the type of toxicity of the chemical composition according to the method of claim 1;

and

b) selecting the chemical compositions of unknown toxicity for preclinical animal studies that have a potential correlation with chemical compounds that are less toxic.

13. The method of claim 12 wherein the predicting is by high throughput screening.

14. The method of claim 1 wherein the molecular profiles of the chemical compositions having known liver toxicity comprise molecular profiles of chemical compositions of two or more different the types of toxicities.

15. A method of generating a library of molecular profiles wherein each molecular profiles is from a population of LSCs contacted with a chemical composition having a known liver toxicity, wherein the molecular profiles are created by the method comprising

a) contacting two or more isolated populations of LSCs with two or more chemical compositions composition having known liver toxicity, wherein each population of LSCs is contacted with one chemical composition

b) creating a molecular profile identifying the alterations in gene expression in the population of LSCs contacted with the chemical compositions compared to LSCs in contact with culture medium only

c) identifying sets of commonly altered RNAs of step b) within molecular profiles from LSCs contacted with chemical compounds of similar type of known toxicity

d) compiling a library of the molecular profiles;

wherein the library is in a computer readable media for use in a computer.

16. The method of claim 15, wherein the library comprises molecular profiles for at least 20 chemical compositions.

17. A systematic method of predicting the type of toxicity of a chemical composition of unknown toxicity, the method comprising the steps of:

a) contacting an isolated population of mammalian LSCs with the chemical compound of unknown toxicity

b) creating a molecular profile identifying the alterations in gene expression in the population of LSCs contacted with the chemical compositions compound of unknown toxicity compared to LSCs in contact with culture medium only; and

c) comparing the molecular profile of step b) with the library of molecular profiles of LSCs contacted with chemical compositions having known liver toxicities generated by the method of claim 15;

wherein the type of toxicity of the chemical composition of unknown toxicity is predicted by correlating common alterations in molecular profiles from LSCs contacted with the chemical compositions having known liver toxicities and unknown toxicities identified from the comparison of step c).

18. The method of claim 17, wherein the chemical composition of unknown toxicity is a known or an unknown chemical compound.

19. The method of claim 17, further wherein the LSCs are of human origin.

20. The method of claim 19, further wherein the chemical compositions of known toxicities are selected from the group consisting of agents that are toxic to cells of one or more reproductive organs, teratogenic agents and carcinogens.

21. The method of claim 19, further wherein the chemical compositions of known toxicities are selected from the group consisting of agricultural chemicals, cosmetics, and environmental contaminants.

22. The method of claim 17, further wherein the LSCs are of non-human mammalian origin.

23. The method of claim 22, wherein the non-human mammals are rodents.

24. The method of claim 22, further wherein the chemical compositions of known toxicities are selected from the group consisting of agents that are toxic to cells of one or more reproductive organs, teratogenic agents and carcinogens.

25. The method of claim 22, further wherein the chemical compositions of known toxicities are selected from the group consisting of agricultural chemicals, cosmetics, and environmental contaminants.

26. A method of screening chemical compositions for liver toxicity preclinical animal studies, said method comprising

a) predicting the type of toxicity of the chemical composition according to the method of claim 17;

and

b) selecting the chemical compositions of unknown toxicity for preclinical animal studies that have a potential correlation with chemical compounds that are less toxic.

27. The method of claim 26 wherein the predicting is by high throughput screening.